Investigation of dual modification on physicochemical, morphological, thermal, pasting, and retrogradation characteristics of sago starch

Abstract The aim of this study was to evaluate the characteristics of dually modified sago starch by acid hydrolysis (AH)‐hydroxypropylation (HP). For this purpose, sago starch was modified with the combination by AH (5–20 h hydrolysis times) followed by HP (5%–25% ratio of propylene oxide) processes. The results showed that the dual modification of the sago starch structure didn't have a significant effect on the size of starch granules, and the granule size was in the range of 0.005–0.151 µm; however, the pasting properties and the glass transition temperature decreased significantly (p < .05). Increasing the level of propylene oxide from 5% to 25% caused a significant increase in the substitution degree (DS) and swelling ability of starches and reduced the syneresis, while with increasing acid hydrolysis time from 5 h to 20 h, starch swelling decreased and syneresis increased (p < .05). AH process at high hydrolysis times (20 h) increased the gelatinization temperatures and decreased retrogradation temperatures. Increasing the level of propylene oxide in both single and dual modification reduced the temperatures and enthalpy of gelatinization and retrogradation of sago starch. In summary, dually modified sago starch has a great potential to use in specific food products such as frozen dough or frozen bakery products.


| INTRODUC TI ON
Starch is a major and noticeable storage polysaccharide in plant sources that consists of two parts, including amylose (the linear polymer) and amylopectin (the highly branched polymer) (Sondari et al., 2021). Starch is an abundant, available, cheap, and biodegradable polysaccharide found in different parts of plants such as fruits, leaves, roots, flowers, seeds, and stems, which it uses as an important source of energy and carbon. Cereals, roots, tubers, legumes, and some unripe and green fruits such as mangos and bananas are major sources of starch. Starches in various plant sources have different and unique sizes, shapes, compositions, and structures (Ashogbon, 2021; Krithika & Ratnamala, 2019).
The sago plant (Metroxylone sagu Rottb) is one of the sources of starch in the world (Zailani et al., 2021). This starch contains 27% amylose and 73% amylopectin and is mainly grown and exported in Southeast Asia (Ekramian et al., 2021;Xue Mei et al., 2020). Sago starch in its natural form has several disadvantages such as low solubility in cold water, formation of chewy and opaque paste, spoilage during storage, syneresis, easy retrogradation, lack of emulsifying property, and so on, which limit its use in various food products and food industry (Zainal Abiddin et al., 2018). The modification process is often used to improve the application and functionality of starches, which is performed by physical, chemical, genetic, or enzymatic methods alone or in combination (Hartiningsih et al., 2020).
The chemical modification process of starch structure involves the introduction of different functional groups (such as ester, carboxylic, ether, and amino groups) into the structure of starch molecules without changing the size and shape of molecules, which causes significant changes in the physicochemical and functional properties of starch. For example, modifying the structure of native starches improves their behaviors in terms of retrogradation, pasting, and gelatinization (Hong et al., 2020). The hydroxypropylation process is a chemical modification method in which hydrophilic bulky groups of hydroxyl propylene introduce to the structure of starch, thereby increasing the solubility, cohesiveness, paste clarity, enzymatic digestibility, and freeze-thaw stability of the starch.
Because hydroxypropylated starches have enhanced characteristics, they are used in food products, pharmaceutical capsules, and biodegradable films (Chen et al., 2021). One of the most widely used methods to modify the structure of starch is acid hydrolysis, in which different dilute acids (H 2 SO 4 , HCl, or H 3 PO 3 ) are used to treat the starch slurry under temperature lower than the gelatinization temperature (40-60°C) . This chemical method is inexpensive and can change the microstructure, crystalline, viscoelastic, gelatinization, and digestibility characteristics of starch (Aminian et al., 2013).
The dual modification process often improves the functionality of starches better than a single modification. Homogeneous and heterogeneous methods are used for dual modification of starches.
In the initial type (homogeneous), two similar methods are used, for example, two physical or two chemical methods. However, in the second type (heterogeneous), two different methods are used, such as combining the physical and chemical methods (Ashogbon, 2021).
Previous research has shown that the combined use of hydroxypropylation and acid hydrolysis processes (dual modification) improves the properties and performance of starches, and these chemical processes have a synergistic effect (Fouladi & Mohammadi Nafchi, 2014;Javadian et al., 2021;Li et al., 2018).

| Materials
Sago starch and propylene oxide were purchased from SIM Supply Company Sdn. Bhd. and Sigma Aldrich Company, respectively. Other chemicals used in this research were prepared by Merck Company and were based on analytical grade.

| Preparation of dual modified sago starch
To prepare the dual modified sago starch, first acid hydrolysis (AH) was done by hydrochloric acid, and then hydroxypropylation (HP) of the hydrolyzed starch was performed. The sago starch slurry was prepared by mixing starch (400 g; based on dry weight) with a dilute solution of hydrochloric acid (0.14 N) at 50°C to reach a final weight of 1000 g. While stirring (at 200 rpm), the starch slurry was incubated at 50°C for 5, 10, 15, and 20 h to prepare hydrolyzed sago starch with various molecular weights. After that, the pH of starch suspensions was reached 5.5 by the addition of 1% sodium hydroxide. The starch samples were then washed with distilled water and filtered through Whatman No. 4 filter paper. The hydrolyzed sago starches were kept at 40°C overnight in an oven to dry completely (Abdorreza et al., 2012). To adding hydroxypropyl group to hydrolyzed sago starch, the hydrolyzed starch solution (20% w/v) was mixed with sodium sulfate (20% w/v) and stirred.
The pH of the starch suspension was reached above 10.5 with the addition of 5% sodium hydroxide. Different proportions (5, 15, and 25%; based on the dry starch weight) of propylene oxide (as etherifying agent) was added to the starch suspension. After that, the starch samples were capped and stirred at 200 rpm for 30 min at room temperature. The pH of starch suspensions was then reached 5.5 using 10% HCl. The dual modified starch samples were washed with distilled water until all sulfate was removed. The modified starches were heated in an oven (at 40°C) until they reached a moisture content of 10%. After the drying process, the starches were ground and then passed through the sieve with meshes of 250 µm (Aminian et al., 2013).

| Scanning electron microscopy (SEM)
The morphology or microstructure of the sago starch granules was determined using SEM (Leica Cambridge). For this purpose, 1 g of starch was coated with a layer of gold/palladium and transferred to the SEM and finally observed at 20 kV (Majzoobi et al., 2012).

| The molar substitution determination (MS)
100 mg of starch was mixed with 25 ml of H 2 SO 4 (0.5 M) and then heated at 60°C in a water bath to dissolve the starch sample. The starch solution was cooled at room temperature and diluted with distilled water (to a volume of 100 ml). The starch solution (1 ml) was transferred into graduated test tubes (25 ml) and immersed in an ice bath, and concentrated H 2 SO 4 (8 ml) was incorporated dropwise.
After complete stirring, the tubes were kept for 20 min in a hot water bath and then transferred to an ice bath. 0.6 ml of ninhydrin reagent solution (3% ninhydrin in 5% Na 2 S 2 O 5 ) was incorporated into the solutions. After shaking, the tubes were placed in a hot water bath at 25°C for 100 min. The volume of solution was reached 25 ml with the addition of concentrated H 2 SO 4 and stirred and kept still for 10 min.

| The water solubility and swelling determination
1 g of starch was transferred to a plastic centrifuge tube (50 ml) and mixed with distilled water (30 ml). While stirring, the tube was heated in a hot water bath at 95°C for 30 min. The sample was then cooled with cold water to reach room temperature and then centrifuged for 15 min at 700 × g. The resulting supernatant was transferred to a container and dried (at 120°C for 4 h). Finally, the starch sample was weighed, and its water solubility and swelling amounts were obtained through the following equations (Trela et al., 2020):

| The pasting characteristics determination
Alternation in the starch slurry viscosity during heating processing was studied with a rapid viscosity analyzer (RVA). The starch sample (4 g) was mixed with distilled water (25 g). The initial desired speed and the speed of the resting stage were 960 rpm and 160 rpm, respectively. At first, the starch sample was kept at 50°C for 1 min, and then its temperature increased rapidly to 95°C (at a speed of 14°C/min) and kept at this temperature for about 5 min.
After that, the sample was cooled to 50°C and kept for 2 min at this temperature. In this experiment, pasting temperature, time to reach peak viscosity, peak, and final viscosity were studied (Karim et al., 2008).

| The gelatinization properties and glass transition temperature evaluation
The thermal characteristics of the starches were measured using the differential scanning calorimeter (DSC). The starch suspension in distilled water (in a ratio of 3:1 w/w) was prepared and weighed in the steel container of the DSC device and kept for 24 h at room temperature to reach equilibrium. The container containing the starch suspension was then transferred into the DSC device and heated and scanned at 10-115°C. A DSC container without starch suspension was used as a control. The thermal characteristics of starch samples including onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and gelatinization enthalpy (ΔH) were calculated from the DSC curves (Majzoobi & Beparva, 2013). The glass transition temperature (Tg) was considered as the middle point between the beginning and the end of the curve changes in the heat flow.

| The starch retrogradation investigation
To investigate the retrogradation of starch samples, the heated samples in the DSC test were kept in the refrigerator (4 ± 1°C) for 3 weeks, and then their onset temperature (To), peak temperature (Tp), final temperature (Tf), and gelatinization enthalpy (ΔH) were measured by the DSC device (Ruales & Nair, 1994;Yusnita et al., 2017).

| The syneresis determination
To determine the syneresis of starch gel, the starch suspension (5% w/v) was prepared and heated for 30 min at 90°C. The starch suspension was cooled in an ice-water bath to room temperature and then centrifuged at 3200 rpm for 15 min. Finally, the syneresis of starch gel was reported as a percentage of the amount of water separated after centrifugation (Sodhi & Singh, 2005).

| Statistical analysis
Statistical analysis of data obtained from the experiments was done using IBM SPSS Statistics 22.0. One-way analysis of variance (one-way ANOVA) followed by Duncan multirange post hoc test was used to compare means at p < .05 significance level among different samples. Swelling (g∕g dried starch) = sediment weight inside the centrifuge tube dry starch weight caused partial destruction of the modified starch structure and increased the depressions in the surface of the granules, and also increased the accumulation of starch granules. Sago starch granules generally had an irregular round shape.

| Morphology and granular size
Granular size is a major factor in the reactions of particles with each other, mixing, and homogenization in food product formulations (Riley et al., 2008). It is generally known that small-and medium-sized starch granules have different applications in the food industry (Omojola et al., 2010). Figure (Gunaratne & Corke, 2007). The increased water solubility of various starches due to acid hydrolysis and hydroxypropylation process has also been observed by other researchers (Biduski et al., 2017;Hartiningsih et al., 2020;Koksel et al., 2008;Li et al., 2020;Wattanachant et al., 2003).  (John et al., 2002). However, at constant times of hydrolysis, with increasing propylene oxide ratio from 5% to 25%, the swelling of starches significantly increased (p < .05). This increase is probably due to the fact that during the hydroxypropylation process, the starch structure weakens and the water penetration into the granules increases, as a result of which the water absorption capacity of the starch granules increases and their swelling power increases (Singh et al., 2004). In general, the lowest swelling rate was observed in HP5AH20 (1.

| The gelatinization properties and glass transition temperature
During heating of starch granules in the presence of excess water, crystalline structures, which are mainly composed of amylopectin branched structures, are converted into amorphous sections during an endothermic process. The energy required to convert crystal structures of starch into amorphous parts is determined by a differential calorimetric test. Gelatinization temperature refers to the temperature at which the crystal structures of the starch granules gradually melt to an amorphous form. Table 2   Note: The values are mean ±SE (n = 3). Different letters show significant differences at 5% level of probability between values in the same columns. NSS, Native sago starch; the numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (hours) of acid hydrolysis. Note: The values are mean ±SE (n = 3). Different letters show significant differences at 5% level of probability between values in the same columns. NSS, Native sago starch; the numbers after "HP' represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (h) of acid hydrolysis.
In general, acid hydrolysis and hydroxypropylation reduced the order of sago starch structure and thus significantly reduced the Tg compared to native sago starch (p < .05). Decreased Tg of modified starches due to acid hydrolysis and hydroxypropylation process has been reported by other researchers (Chatakanonda et al., 2011;Chotipratoom et al., 2015;Omojola et al., 2010;Yeh & Yeh, 1993).

F I G U R E 6
Comparison of Tg values (°C) of native and dually modified sago starch samples by acid hydrolysis-hydroxypropylation. Bars represent mean (n = 3) ± SD. Different letters on the bars indicate a significant difference at 5% level of probability among starch samples. NSS, Native sago starch; the numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (h) of acid hydrolysis Note: The values are mean ±SE (n = 3). Different letters show significant differences at 5% level of probability between values in the same columns. NSS: Native sago starch; the numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (h) of acid hydrolysis.

TA B L E 3
The retrogradation properties of the native and dual modified sago starches

| The retrogradation of starch
Changes in starch during gelatinization and retrogradation are important to evaluate its functional properties for food processing, during digestion and in industrial applications (Chang et al., 2021). These characteristics evaluate the acceptance quality, shelf life, and nutritional value of the final products (Wang & Copeland, 2013). Retrogradation of starch often has adverse effects because it participates in the process of staling bread and other starch-rich foods, reducing the shelf life and acceptance of products and producing a lot of waste. When starch is heated in the presence of water and then cooled, the amylose and amylopectin chains are broken down into new structures called starch retrogradation (Hoover et al., 2010). Table 3  In the process of hydroxypropylation, with the entry of hydroxypropyl groups into the starch chains, the destruction of intermolecular and intramolecular bonds occurs, and thus the starch structure is weakened. As a result, the mobility of the starch chains in the amorphous regions increases. In this way, the retrogradation of starch is reduced (Olayinka et al., 2015). Since there are two different pathways for the effect of the acid hydrolysis process on starch retrogradation, the effects of this process are different under various conditions. On the one hand, the removal of α (1→6) branching points in the amylopectin and amylose hydrolysis occurs due to the acid hydrolysis, which can increase the retrogradation intensity of starch gels. On the other hand, the small molecules in the residual acidic hydrolyzed starch have an irregular effect on the recrystallization of the hydrolyzed starch gels and demonstrate the opposite effect (Wang et al., 2015). Similarly, previous research has shown that the hydroxypropylation of different starches reduced the tendency of starch samples to retrograde (Chotipratoom et al., 2015;Lawal et al., 2008;Oh et al., 2019;Senanayake et al., 2014). Kang et al. (1997) found that due to acid hydrolysis and increasing the time of this process, retrogradation of rice starch increased. However, Gunaratne and Corke (2007) reported that acid hydrolysis had no significant effect on the retrogradation of corn and potato starches but reduced the intensity of retrogradation in wheat starch. There are generally conflicting reports on the effect of the acid hydrolysis process on the rate of starch retrogradation (Wang et al., 2015).

| Syneresis
The stability of starch pastes can be shown by their syneresis values. Syneresis indicates the amount of water that separates from the starch granules after the freezing-thawing cycle or during the storage period (Wang et al., 2015). Higher values of syneresis indicate less stability of starch gel and a higher rate of starch retrogradation (Liu et al., 2014). Indirectly, the retrogradation and syneresis properties of starches are influenced by the structural arrangement of starch chains in the amorphous and crystalline regions of the starch granules (Kaur et al., 2007).
The syneresis percentage of native and dual modified sago starches by acid hydrolysis-hydroxypropylation is compared in Figure 7, which shows that at constant acid hydrolysis time, by increasing the propylene oxide ratio from 5% to 25%, the syneresis value of modified starch significantly decreased (p < .05). However, at a constant ratio of propylene oxide, increasing the acid hydrolysis time from 5 to 20 h led to a significant increase in the percentage of syneresis (p < .05). The highest and lowest syneresis values were observed in HP5AH20 (4.65%) and HP25AH5 (1.61%), respectively.
The syneresis is due to rearrangement and subsequent crosslinking between the amylopectin and amylose, but because the hydroxypropyl groups attached to the amylopectin and amylose during the hydroxypropylation process create a spatial barrier, this barrier prevents the chains from coming too close to each other during storage and reduces syneresis (Kaur et al., 2007). The hydrophilic hydroxypropyl groups can increase the water holding capacity of starch molecules and reduce syneresis in modified starches. The higher syneresis of starch samples due to the increase in acid hydrolysis time is probably due to the reduction of water holding capacity and water absorption of acid hydrolyzed starches compared to the native starch. A remarkable effect of hydroxypropylation process in reducing the syneresis tendency of various starches has been reported in several scientific studies (Kaur et al., 2007;Lawal et al., 2008;Lee & Yoo, 2011;Maulani et al., 2019;Senanayake et al., 2014;Shaikh et al., 2017). Increased syneresis of sweet potato, banana, and wheat starches gels due to the acid hydrolysis process has also been observed by Kaur et al. (2011).

| CON CLUS ION
In this study, it is observed that the dual modification of sago starch structure by acid hydrolysis-hydroxypropylation improves the starch water solubility. Dual modification of starches at low acid hydrolysis time and high ratio of propylene oxide led to a remarkable reduction in the retrogradation intensity and syneresis percentage of modified starches compared to native sago starch. Due to the dual modification, the structure of sago starch became more irregular, and the peak viscosity occurred in less time. The results of this study generally demonstrated that the dual modified sago starch, especially low acid hydrolysis time and high ratio of propylene oxide, can be used in the food industry to reduce the staling rate and syneresis of food products.

ACK N OWLED G EM ENTS
We thank Prof. Fazilah Ariffin from Universiti Sains Malaysia for her valuable input and critical reading of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author, upon reasonable request. Bars represent mean (n = 3) ± SD. Different letters on the bars indicate a significant difference at 5% level of probability among starch samples. NSS, Native sago starch; the numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (h) of acid hydrolysis